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<title>Combined or independent SuperCell runs for different samples</title>

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<h1 class="title toc-ignore">Combined or independent SuperCell runs for different samples</h1>



<p>Comparing a combined (i.e., processing samples together) and an independent (i.e., processing samples separately) construction of metacells with <em>SuperCell</em> (related to <span class="citation">@daskelly</span> question <a href="https://github.com/GfellerLab/SuperCell/issues/11#issuecomment-1090916447" class="uri">https://github.com/GfellerLab/SuperCell/issues/11#issuecomment-1090916447</a>).</p>
<div class="sourceCode" id="cb1"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb1-1"><a href="#cb1-1" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(SuperCell)</span>
<span id="cb1-2"><a href="#cb1-2" aria-hidden="true" tabindex="-1"></a><span class="fu">library</span>(Matrix)</span>
<span id="cb1-3"><a href="#cb1-3" aria-hidden="true" tabindex="-1"></a><span class="fu">data</span>(cell_lines)</span>
<span id="cb1-4"><a href="#cb1-4" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb1-5"><a href="#cb1-5" aria-hidden="true" tabindex="-1"></a>GE <span class="ot">&lt;-</span> cell_lines<span class="sc">$</span>GE</span>
<span id="cb1-6"><a href="#cb1-6" aria-hidden="true" tabindex="-1"></a>cell.meta <span class="ot">&lt;-</span> cell_lines<span class="sc">$</span>meta</span></code></pre></div>
<div class="sourceCode" id="cb2"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb2-1"><a href="#cb2-1" aria-hidden="true" tabindex="-1"></a>gamma <span class="ot">&lt;-</span> <span class="dv">20</span> <span class="co"># graining level</span></span>
<span id="cb2-2"><a href="#cb2-2" aria-hidden="true" tabindex="-1"></a>n.pc  <span class="ot">&lt;-</span> <span class="dv">10</span> <span class="co"># number of PCs</span></span></code></pre></div>
<p>To compare results, we use 2 samples that correspond to two different cancer cell lines (data from <a href="https://doi.org/10.1038/s41592-019-0425-8">Tian et al., 2019</a>)</p>
<div class="sourceCode" id="cb3"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb3-1"><a href="#cb3-1" aria-hidden="true" tabindex="-1"></a>cell.idx.HCC827 <span class="ot">&lt;-</span> <span class="fu">which</span>(cell.meta <span class="sc">==</span> <span class="st">&quot;HCC827&quot;</span>)</span>
<span id="cb3-2"><a href="#cb3-2" aria-hidden="true" tabindex="-1"></a>cell.idx.H838   <span class="ot">&lt;-</span> <span class="fu">which</span>(cell.meta <span class="sc">==</span> <span class="st">&quot;H838&quot;</span>)</span></code></pre></div>
<div id="a-combined-analysis-construction-of-metacells-processing-two-samples-together" class="section level2">
<h2>A combined analysis – construction of metacells processing two samples together</h2>
<div class="sourceCode" id="cb4"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb4-1"><a href="#cb4-1" aria-hidden="true" tabindex="-1"></a>SC.HCC827.H838 <span class="ot">&lt;-</span> <span class="fu">SCimplify</span>(</span>
<span id="cb4-2"><a href="#cb4-2" aria-hidden="true" tabindex="-1"></a>  GE[,<span class="fu">c</span>(cell.idx.HCC827, cell.idx.H838)],  <span class="co"># log-normalized gene expression matrix</span></span>
<span id="cb4-3"><a href="#cb4-3" aria-hidden="true" tabindex="-1"></a>  <span class="at">gamma =</span> gamma, <span class="co"># graining level</span></span>
<span id="cb4-4"><a href="#cb4-4" aria-hidden="true" tabindex="-1"></a>  <span class="at">cell.split.condition =</span> cell.meta[<span class="fu">c</span>(cell.idx.HCC827, cell.idx.H838)], <span class="co"># metacell do not mix cells from different cell lines</span></span>
<span id="cb4-5"><a href="#cb4-5" aria-hidden="true" tabindex="-1"></a>  <span class="at">n.pc =</span> n.pc) <span class="co"># number of proncipal components to use</span></span>
<span id="cb4-6"><a href="#cb4-6" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-7"><a href="#cb4-7" aria-hidden="true" tabindex="-1"></a>genes.use <span class="ot">&lt;-</span> SC.HCC827.H838<span class="sc">$</span>genes.use</span>
<span id="cb4-8"><a href="#cb4-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-9"><a href="#cb4-9" aria-hidden="true" tabindex="-1"></a>SC.HCC827.H838<span class="sc">$</span>cell.line <span class="ot">&lt;-</span> <span class="fu">supercell_assign</span>(cell.meta[<span class="fu">c</span>(cell.idx.HCC827, cell.idx.H838)], </span>
<span id="cb4-10"><a href="#cb4-10" aria-hidden="true" tabindex="-1"></a>                                             <span class="at">supercell_membership =</span> SC.HCC827.H838<span class="sc">$</span>membership)</span>
<span id="cb4-11"><a href="#cb4-11" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-12"><a href="#cb4-12" aria-hidden="true" tabindex="-1"></a>SC.GE.HCC827.H838 <span class="ot">&lt;-</span> <span class="fu">supercell_GE</span>(GE[,<span class="fu">c</span>(cell.idx.HCC827, cell.idx.H838)], <span class="at">groups =</span> SC.HCC827.H838<span class="sc">$</span>membership)</span>
<span id="cb4-13"><a href="#cb4-13" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-14"><a href="#cb4-14" aria-hidden="true" tabindex="-1"></a>SC.HCC827.H838<span class="sc">$</span>SC_PCA <span class="ot">&lt;-</span> <span class="fu">supercell_prcomp</span>(</span>
<span id="cb4-15"><a href="#cb4-15" aria-hidden="true" tabindex="-1"></a>  Matrix<span class="sc">::</span><span class="fu">t</span>(SC.GE.HCC827.H838),</span>
<span id="cb4-16"><a href="#cb4-16" aria-hidden="true" tabindex="-1"></a>  <span class="at">supercell_size =</span> SC.HCC827.H838<span class="sc">$</span>supercell_size, </span>
<span id="cb4-17"><a href="#cb4-17" aria-hidden="true" tabindex="-1"></a>  <span class="at">genes.use =</span> genes.use)</span>
<span id="cb4-18"><a href="#cb4-18" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-19"><a href="#cb4-19" aria-hidden="true" tabindex="-1"></a>SC.HCC827.H838<span class="sc">$</span>SC_UMAP <span class="ot">&lt;-</span> <span class="fu">supercell_UMAP</span>(</span>
<span id="cb4-20"><a href="#cb4-20" aria-hidden="true" tabindex="-1"></a>  SC.HCC827.H838, </span>
<span id="cb4-21"><a href="#cb4-21" aria-hidden="true" tabindex="-1"></a>  <span class="at">n_neighbors =</span> <span class="dv">10</span>)</span>
<span id="cb4-22"><a href="#cb4-22" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb4-23"><a href="#cb4-23" aria-hidden="true" tabindex="-1"></a><span class="fu">supercell_plot_UMAP</span>(</span>
<span id="cb4-24"><a href="#cb4-24" aria-hidden="true" tabindex="-1"></a>    SC.HCC827.H838,</span>
<span id="cb4-25"><a href="#cb4-25" aria-hidden="true" tabindex="-1"></a>    <span class="at">group =</span> <span class="st">&quot;cell.line&quot;</span>,</span>
<span id="cb4-26"><a href="#cb4-26" aria-hidden="true" tabindex="-1"></a>    <span class="at">title =</span> <span class="fu">paste0</span>(<span class="st">&quot;Combined construction of HCC827 and H838 metacells&quot;</span>)</span>
<span id="cb4-27"><a href="#cb4-27" aria-hidden="true" tabindex="-1"></a>  )</span></code></pre></div>
<p><img src="" /><!-- --></p>
</div>
<div id="independent-analysis-construction-of-metacells-for-each-sample-independently-applying-the-same-granularity-gamma-and-the-same-set-of-features-genes.use" class="section level2">
<h2>Independent analysis – construction of metacells for each sample independently (applying the same granularity <code>gamma</code> and the same set of features <code>genes.use</code>)</h2>
<div class="sourceCode" id="cb5"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb5-1"><a href="#cb5-1" aria-hidden="true" tabindex="-1"></a>SC.HCC827 <span class="ot">&lt;-</span> <span class="fu">SCimplify</span>(GE[,cell.idx.HCC827],  <span class="co"># log-normalized gene expression matrix</span></span>
<span id="cb5-2"><a href="#cb5-2" aria-hidden="true" tabindex="-1"></a>                <span class="at">gamma =</span> gamma, <span class="co"># graining level</span></span>
<span id="cb5-3"><a href="#cb5-3" aria-hidden="true" tabindex="-1"></a>                <span class="at">n.pc =</span> n.pc, <span class="co"># number of proncipal components to use</span></span>
<span id="cb5-4"><a href="#cb5-4" aria-hidden="true" tabindex="-1"></a>                <span class="at">genes.use =</span> genes.use) <span class="co"># using the same set of genes as for the combined analysis</span></span>
<span id="cb5-5"><a href="#cb5-5" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; Warning in SCimplify(GE[, cell.idx.HCC827], gamma = gamma, n.pc = n.pc, : Normal</span></span>
<span id="cb5-6"><a href="#cb5-6" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; pca is computed because number of cell is low for irlba::irlba()</span></span>
<span id="cb5-7"><a href="#cb5-7" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-8"><a href="#cb5-8" aria-hidden="true" tabindex="-1"></a>SC.HCC827<span class="sc">$</span>cell.line <span class="ot">&lt;-</span> <span class="fu">supercell_assign</span>(cell.meta[cell.idx.HCC827], <span class="at">supercell_membership =</span> SC.HCC827<span class="sc">$</span>membership)</span>
<span id="cb5-9"><a href="#cb5-9" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-10"><a href="#cb5-10" aria-hidden="true" tabindex="-1"></a>SC.H838 <span class="ot">&lt;-</span> <span class="fu">SCimplify</span>(GE[,cell.idx.H838],  <span class="co"># log-normalized gene expression matrix</span></span>
<span id="cb5-11"><a href="#cb5-11" aria-hidden="true" tabindex="-1"></a>                <span class="at">gamma =</span> gamma, <span class="co"># graining level</span></span>
<span id="cb5-12"><a href="#cb5-12" aria-hidden="true" tabindex="-1"></a>                <span class="at">n.pc =</span> n.pc, <span class="co"># number of proncipal components to use</span></span>
<span id="cb5-13"><a href="#cb5-13" aria-hidden="true" tabindex="-1"></a>                <span class="at">genes.use =</span> genes.use) <span class="co"># using the same set of genes as for the combined analysis</span></span>
<span id="cb5-14"><a href="#cb5-14" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; Warning in SCimplify(GE[, cell.idx.H838], gamma = gamma, n.pc = n.pc, genes.use</span></span>
<span id="cb5-15"><a href="#cb5-15" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; = genes.use): Normal pca is computed because number of cell is low for</span></span>
<span id="cb5-16"><a href="#cb5-16" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; irlba::irlba()</span></span>
<span id="cb5-17"><a href="#cb5-17" aria-hidden="true" tabindex="-1"></a>SC.H838<span class="sc">$</span>cell.line <span class="ot">&lt;-</span> <span class="fu">supercell_assign</span>(cell.meta[cell.idx.H838], <span class="at">supercell_membership =</span> SC.H838<span class="sc">$</span>membership)</span>
<span id="cb5-18"><a href="#cb5-18" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-19"><a href="#cb5-19" aria-hidden="true" tabindex="-1"></a>SC.merged <span class="ot">&lt;-</span> <span class="fu">supercell_merge</span>(<span class="fu">list</span>(SC.HCC827, SC.H838), <span class="at">fields =</span> <span class="fu">c</span>(<span class="st">&quot;cell.line&quot;</span>))</span>
<span id="cb5-20"><a href="#cb5-20" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-21"><a href="#cb5-21" aria-hidden="true" tabindex="-1"></a><span class="co"># compute metacell gene expression for SC.HCC827</span></span>
<span id="cb5-22"><a href="#cb5-22" aria-hidden="true" tabindex="-1"></a>SC.GE.HCC827 <span class="ot">&lt;-</span> <span class="fu">supercell_GE</span>(GE[, cell.idx.HCC827], <span class="at">groups =</span> SC.HCC827<span class="sc">$</span>membership)</span>
<span id="cb5-23"><a href="#cb5-23" aria-hidden="true" tabindex="-1"></a><span class="co"># compute metacell gene expression for SC.H838</span></span>
<span id="cb5-24"><a href="#cb5-24" aria-hidden="true" tabindex="-1"></a>SC.GE.H838 <span class="ot">&lt;-</span> <span class="fu">supercell_GE</span>(GE[, cell.idx.H838], <span class="at">groups =</span> SC.H838<span class="sc">$</span>membership)</span>
<span id="cb5-25"><a href="#cb5-25" aria-hidden="true" tabindex="-1"></a><span class="co"># merge GE matricies</span></span>
<span id="cb5-26"><a href="#cb5-26" aria-hidden="true" tabindex="-1"></a>SC.GE.merged <span class="ot">&lt;-</span> <span class="fu">supercell_mergeGE</span>(<span class="fu">list</span>(SC.GE.HCC827, SC.GE.H838))</span>
<span id="cb5-27"><a href="#cb5-27" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; 2000 of common genes has been detected</span></span>
<span id="cb5-28"><a href="#cb5-28" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-29"><a href="#cb5-29" aria-hidden="true" tabindex="-1"></a>SC.merged<span class="sc">$</span>SC_PCA <span class="ot">&lt;-</span> <span class="fu">supercell_prcomp</span>(</span>
<span id="cb5-30"><a href="#cb5-30" aria-hidden="true" tabindex="-1"></a>  Matrix<span class="sc">::</span><span class="fu">t</span>(SC.GE.merged),</span>
<span id="cb5-31"><a href="#cb5-31" aria-hidden="true" tabindex="-1"></a>  <span class="at">supercell_size =</span> SC.merged<span class="sc">$</span>supercell_size, </span>
<span id="cb5-32"><a href="#cb5-32" aria-hidden="true" tabindex="-1"></a>  <span class="at">genes.use =</span> genes.use)</span>
<span id="cb5-33"><a href="#cb5-33" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-34"><a href="#cb5-34" aria-hidden="true" tabindex="-1"></a>SC.merged<span class="sc">$</span>SC_UMAP <span class="ot">&lt;-</span> <span class="fu">supercell_UMAP</span>(</span>
<span id="cb5-35"><a href="#cb5-35" aria-hidden="true" tabindex="-1"></a>  SC.merged, </span>
<span id="cb5-36"><a href="#cb5-36" aria-hidden="true" tabindex="-1"></a>  <span class="at">n_neighbors =</span> <span class="dv">10</span>)</span>
<span id="cb5-37"><a href="#cb5-37" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb5-38"><a href="#cb5-38" aria-hidden="true" tabindex="-1"></a>g <span class="ot">&lt;-</span> <span class="fu">supercell_plot_UMAP</span>(</span>
<span id="cb5-39"><a href="#cb5-39" aria-hidden="true" tabindex="-1"></a>    SC.merged,</span>
<span id="cb5-40"><a href="#cb5-40" aria-hidden="true" tabindex="-1"></a>    <span class="at">group =</span> <span class="st">&quot;cell.line&quot;</span>,</span>
<span id="cb5-41"><a href="#cb5-41" aria-hidden="true" tabindex="-1"></a>    <span class="at">title =</span> <span class="fu">paste0</span>(<span class="st">&quot;Independent construction of HCC827 and H838 metacells&quot;</span>)</span>
<span id="cb5-42"><a href="#cb5-42" aria-hidden="true" tabindex="-1"></a>  )</span></code></pre></div>
<p><img src="" /><!-- --></p>
</div>
<div id="combined-and-independent-analyses-do-not-result-in-the-same-metacell-construction." class="section level2">
<h2>Combined and independent analyses do not result in the same metacell construction.</h2>
<p>As the dimensionality reductions (even on the same set of features) are different for the combined (<em>HCC827</em>+<em>H838</em>) dataset and for the independent (<em>HCC827</em> and <em>H838</em> separately) datasets. The first PCA basen on <em>global</em> variability between two cell lines and the PCAs from the second approach represent <em>local</em> variability within each cell line. (sample).</p>
<div class="sourceCode" id="cb6"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb6-1"><a href="#cb6-1" aria-hidden="true" tabindex="-1"></a><span class="fu">heatmap</span>(<span class="fu">as.matrix</span>(<span class="fu">table</span>(SC.merged<span class="sc">$</span>membership, SC.HCC827.H838<span class="sc">$</span>membership)), <span class="at">scale =</span> <span class="st">&quot;none&quot;</span>)</span></code></pre></div>
<p><img src="" /><!-- --></p>
<p>Metacell size distribution</p>
<div class="sourceCode" id="cb7"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb7-1"><a href="#cb7-1" aria-hidden="true" tabindex="-1"></a><span class="fu">summary</span>(SC.merged<span class="sc">$</span>supercell_size)</span>
<span id="cb7-2"><a href="#cb7-2" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt;    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. </span></span>
<span id="cb7-3"><a href="#cb7-3" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt;    4.00   10.00   17.00   20.05   23.00   98.00</span></span>
<span id="cb7-4"><a href="#cb7-4" aria-hidden="true" tabindex="-1"></a><span class="fu">summary</span>(SC.HCC827.H838<span class="sc">$</span>supercell_size)</span>
<span id="cb7-5"><a href="#cb7-5" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt;    Min. 1st Qu.  Median    Mean 3rd Qu.    Max. </span></span>
<span id="cb7-6"><a href="#cb7-6" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt;    4.00   10.25   17.50   19.78   27.00   68.00</span></span></code></pre></div>
<p>Also, in the combined analysis, the graining level does not mean that each cell line (or sample) will have this particular graining level. For instance, in the combined analysis, the graining level for <em>HCC827</em> is <code>18.6</code> and for <em>H838</em> is <code>21.8</code>, but this difference might be even more prominent if the heterogeneity and complexity of two samples are more different.</p>
<div class="sourceCode" id="cb8"><pre class="sourceCode r"><code class="sourceCode r"><span id="cb8-1"><a href="#cb8-1" aria-hidden="true" tabindex="-1"></a><span class="do">## Combined analysis</span></span>
<span id="cb8-2"><a href="#cb8-2" aria-hidden="true" tabindex="-1"></a><span class="co"># actual graining level for H838 cell line</span></span>
<span id="cb8-3"><a href="#cb8-3" aria-hidden="true" tabindex="-1"></a><span class="fu">length</span>(cell.idx.H838)<span class="sc">/</span><span class="fu">sum</span>(SC.HCC827.H838<span class="sc">$</span>cell.line <span class="sc">==</span> <span class="st">&quot;H838&quot;</span>)</span>
<span id="cb8-4"><a href="#cb8-4" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; [1] 19.46667</span></span>
<span id="cb8-5"><a href="#cb8-5" aria-hidden="true" tabindex="-1"></a><span class="co"># actual graining level for H838 cell line</span></span>
<span id="cb8-6"><a href="#cb8-6" aria-hidden="true" tabindex="-1"></a><span class="fu">length</span>(cell.idx.HCC827)<span class="sc">/</span><span class="fu">sum</span>(SC.HCC827.H838<span class="sc">$</span>cell.line <span class="sc">==</span> <span class="st">&quot;HCC827&quot;</span>)</span>
<span id="cb8-7"><a href="#cb8-7" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; [1] 20.27586</span></span>
<span id="cb8-8"><a href="#cb8-8" aria-hidden="true" tabindex="-1"></a></span>
<span id="cb8-9"><a href="#cb8-9" aria-hidden="true" tabindex="-1"></a><span class="do">## Independent analysis </span></span>
<span id="cb8-10"><a href="#cb8-10" aria-hidden="true" tabindex="-1"></a><span class="co"># actual graining level for H838 cell line</span></span>
<span id="cb8-11"><a href="#cb8-11" aria-hidden="true" tabindex="-1"></a><span class="fu">length</span>(cell.idx.H838)<span class="sc">/</span><span class="fu">sum</span>(SC.merged<span class="sc">$</span>cell.line <span class="sc">==</span> <span class="st">&quot;H838&quot;</span>)</span>
<span id="cb8-12"><a href="#cb8-12" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; [1] 19.90909</span></span>
<span id="cb8-13"><a href="#cb8-13" aria-hidden="true" tabindex="-1"></a><span class="co"># actual graining level for HCC827 cell line</span></span>
<span id="cb8-14"><a href="#cb8-14" aria-hidden="true" tabindex="-1"></a><span class="fu">length</span>(cell.idx.HCC827)<span class="sc">/</span><span class="fu">sum</span>(SC.merged<span class="sc">$</span>cell.line <span class="sc">==</span> <span class="st">&quot;HCC827&quot;</span>)</span>
<span id="cb8-15"><a href="#cb8-15" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; [1] 20.27586</span></span>
<span id="cb8-16"><a href="#cb8-16" aria-hidden="true" tabindex="-1"></a><span class="co"># actual overall graining level in the combined analysis</span></span>
<span id="cb8-17"><a href="#cb8-17" aria-hidden="true" tabindex="-1"></a><span class="fu">length</span>(SC.merged<span class="sc">$</span>membership)<span class="sc">/</span><span class="fu">max</span>(SC.merged<span class="sc">$</span>membership)</span>
<span id="cb8-18"><a href="#cb8-18" aria-hidden="true" tabindex="-1"></a><span class="co">#&gt; [1] 20.05479</span></span></code></pre></div>
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